Early Alterations of RNA Binding Protein (RBP) Homeostasis and ER Stress-Mediated Autophagy Contributes to Progressive Retinal Degeneration in the rd10 Mouse Model of Retinitis Pigmentosa (RP)

The retinal degeneration 10 (rd10) mouse model is widely used to study retinitis pigmentosa (RP) pathomechanisms. It offers a rather unique opportunity to study trans-neuronal degeneration because the cell populations in question are separated anatomically and the mutated Pde6b gene is selectively expressed in rod photoreceptors. We hypothesized that RNA binding protein (RBP) aggregation and abnormal autophagy might serve as early pathogenic events, damaging non-photoreceptor retinal cell types that are not primarily targeted by the Pde6b gene defect. We used a combination of immunohistochemistry (DAB, immunofluorescence), electron microscopy (EM), subcellular fractionation, and Western blot analysis on the retinal preparations obtained from both rd10 and wild-type mice. We found early, robust increases in levels of the protective endoplasmic reticulum (ER) calcium (Ca2+) buffering chaperone Sigma receptor 1 (SigR1) together with other ER-Ca2+ buffering proteins in both photoreceptors and non-photoreceptor neuronal cells before any noticeable photoreceptor degeneration. In line with this, we found markedly altered expression of the autophagy proteins p62 and LC3, together with abnormal ER widening and large autophagic vacuoles as detected by EM. Interestingly, these changes were accompanied by early, prominent cytoplasmic and nuclear aggregation of the key RBPs including pTDP-43 and FET family RBPs and stress granule formation. We conclude that progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy, along with abnormal cytoplasmic RBP aggregation.


Introduction
Retinitis pigmentosa (RP) is an inherited retinal disease resulting in the progressive degeneration of retinal photoreceptors and ultimately leading to blindness [1,2]. Mutations in

Antibodies
All primary and secondary antibodies and their dilutions used in this study are listed in Table S1. Many of these antibodies have been used by us in both mice and human tissue in previously published studies (see references in Table S1).

rd10 Mouse Model
Studies were performed using retinae from male and female rd10 mice (C57BL/6J-Pde6b rd10/J) [11] and age-matched wild-type (WT) mice (C57BL/6) as controls. It is important to note that the two mouse strains share the same genetic background. The breeding of rd10 mice has been described previously in detail [31]. The animals were kept on a 12 h light/dark cycle with food and water ad libitum. All experiments were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and the German Law for the Protection of Animals and after approval had been obtained by the regulatory authorities of the Forschungszentrum Jülich and the Institute of Laboratory Animal Science (Faculty of Medicine, RWTH Aachen University). A4 application number 11280A4.

Preparation of Retinal Sections and Homogenates
Both rd10 and wild-type mice were deeply anesthetized by isoflurane and sacrificed by decapitation on the following postnatal day: P16, P17, P19, P22, P25, P26, P50 (n = 3 in each age group, otherwise mentioned in the legend). Both the eyeballs were enucleated and used for hematoxylin and eosin staining (H&E), immunohistochemistry, immunofluorescence, transmission electron microscopy (TEM), Western blotting, filter trap assay (FTA), and subcellular fractionation (see also Table S3). For H&E histology, immunohistochemistry, and immunofluorescence analyses, the eyes were cut open at the limbus. The cornea and lens were removed and the retina in the eyecup was fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature. The unfixed eyeballs of WT mice (P26: n = 3) and rd10 (P17: n = 4, P26: n = 3) mice were used for Western blotting. For the filter trap assay, a pool of WT (P26: n = 3) and rd10 (P17: n = 3, P26: n = 3) was used. For the purification of subcellular protein fractions, a Subcellular Protein Fractionation Kit for Tissues (Thermo Scientific, Rockford, IL, USA) was used. Based upon the Western blot analysis performed on these purified protein fractions, the distributions of RBPs in rd10 and age-matched WT mice were determined. Both the left and right retinae of each of rd10-P17 (n = 3), WT-P17 (n = 3) and WT-P26 (n = 3), rd10-P17 (n = 3), and rd10-P26 (n = 3) were collected (see also Table S3). For these analyses, the cornea and lens were removed, and the retina was carefully dissected out of the eyecup for homogenization.

Diaminobenzidine (DAB) Immunohistochemistry
Paraffin sections of 3-4 µm were placed on poly-L-lysine coated slides and allowed to dry in an oven at 37 • C overnight and then processed for immunohistochemistry as described in detail elsewhere [30,32]. The sections were deparaffinized in xylene for 20 min and rehydrated in 100%, 96%, and 70% ethanol for 5 min each, followed by endogenous peroxidase quenching (0.3% H 2 O 2 in methanol) for 20 min. For antigen retrieval, sections were heated in citrate buffer (Dako Target Retrieval solution), pH 6 (Dako, Glostrup, Denmark), for 20 min in a pressure cooker. After washing in PBS, sections were incubated with primary antibody (Table S1) for 1 h at room temperature or 4 • C overnight. After washing in PBS, sections were incubated with appropriate HRP-linked secondary antibodies (ImmunoLogic, Duiven, The Netherlands) for 30 min at room temperature. DAB reagent (ImmunoLogic, Duiven, The Netherlands) was used to visualize antibody binding. The sections were then counter-stained with 6% Mayer's hemalum solution (Merck KGaA, Darmstadt, Germany) for 3 min. All procedures were performed at room temperature. Representative sections formed each eye: WT-P26 (n = 3), rd10-P17 (n = 3), and rd10-P26 (n = 3) were analyzed (see also Table S3).

Immunofluorescence
Single and double immunofluorescence staining were performed as already described [30,32]; representative sections from every genotype were analyzed, (n = 3) WT-P26, (n = 3) rd10-P17, and (n = 3) rd10-P26-and for certain stains, WT-P50 and rd10-P50 were analyzed. Briefly, deparaffinized sections were heated in citrate buffer (Dako Target Retrieval solution), pH 6 (Dako, Glostrup, Denmark) for 20 min in a pressure cooker. Sections were then blocked (to avoid non-specific binding) with ready-to-use 10% normal goat serum (Life Technologies, Frederick, MD, USA) for 1 h at room temperature before incubating with primary antibody at 4 • C overnight. After two washes in TBS-T for 10 min, the sections were incubated with Alexa-conjugated secondary antibody (1:500 in TBS-T) at room temperature for 2 h. Sections were then washed in TBS-T (2 × 10 min) and incubated for 10 min in 0.1% Sudan Black/80% ethanol to suppress endogenous autofluorescence. Finally, the sections were washed for 5 min in TBST and mounted with antifade mounting medium with DAPI (Vectashield with DAPI, H-1200, Vector Laboratories Inc., Burlingame, CA, USA).

Image Acquisition and Semi-Quantitative Analysis
Images of the H&E-stained sections (representative sections from each genotype (n = 3 WT-P17, n = 3 WT-P19, n = 3 WT-P26, n = 3 WT-P50, n = 3 rd10-P17, n = 3 rd10-P19, n = 3 rd10-P26, and n = 3 rd10-P50) and DAB-stained sections (n = 3 WT-P26, n = 3 rd10-P17, and n = 3 rd10-P26) were taken with a Zeiss Axioplan microscope equipped with a 40× objective and an Axio Cam 506 camera. Images from immunofluorescence labeled sections were taken with a Zeiss LSM 700 laser scanning confocal microscope using 20×, 40×, and 63× objectives. Images were acquired by averaging 4 scans per area of interest resulting in an image size of 1024 × 1024 pixels. The laser intensity was kept constant for all of the samples examined. Captured confocal images were analyzed using Adobe Photoshop CS5 and ZEN (Blue edition) 2009 software. Semi-quantitative analysis (See Table S2) was performed manually by examining the sections (one representative section) from each of the three mice from each genotype and assigning the following scores based on the pattern of immunoreactivity (+++ strong immunoreactivity/accumulation, ++ moderate immunoreactivity/accumulation, + mild immunoreactivity, • aggregates, NA, not available/not included in the study).
Semi-quantitative analysis of fluorescence intensity: The average pixel intensity of the target proteins (Calreticulin, SigR1, GRP78, LC3)/field of view were quantified in one representative section from each of three rd10-P17 and rd10-P26 and one representative section each from three age-matched wild type controls of WT-P26. A total of 8-10 random fields at low magnification (20×) were examined, capturing approx. 15-20-30 ROI with a 40× lens from different parts of the retina (quantification was performed only from the ONL and INL). The average background pixel intensity was subtracted. We used the unpaired Student's t-test for comparison between two sample groups. Values represent the mean ± standard deviation (SD). Differences between values were regarded as significant when = (* p <0.05, ** p <0.01).

Western Blot Analysis
Western blots of retina homogenates were performed as described previously [30,32]. Briefly, both the left and right retinae of WT (P26: n = 3) and rd10 mice (P17: n = 4, P26: n = 3) were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Roche Life Science, Penzberg, Germany). The crude lysates were briefly centrifuged and then processed for the bicinchoninic acid protein assay (BCA; Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol. Equal amounts of protein were boiled in Laemmli sample buffer for 5-10 min and processed for SDS-PAGE. The protein gels were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The blots were then blocked with 4% skim milk (Sigma-Aldrich, St. Louis, MO. USA) in TBS-T for 30 min and incubated with a specific primary antibody (Table S1) overnight at 4 • C under agitation. Thereafter, the blots were washed three times in TBS-T for 10 min each and incubated for 1 h with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Scientific). Immunoreactive protein bands were visualized by exposing the blots on an X-ray film (Thermo Scientific, Rockford, IL, USA). Quantification of the band intensities was performed after normalizing to tubulin levels using Adobe Photoshop CS5. Values represent the mean ± SD (* p < 0.05, ** p < 0.01).

Subcellular Fractionation
Briefly, the subcellular protein fractionation kit (Thermo Scientific/Life Technologies) was used to determine subcellular distributions of RNA binding proteins in rd10 and agematched WT mice. Both the left and right retinae of each of WT-P17 (n = 3), rd10-P17 (n = 3) WT-P26 (n = 3), rd10-P17 (n = 3), rd10-P26 (n = 3) were collected, pooled, and homogenized (each group containing a total of 6 retinae). Four subcellular fractions were obtained per sample (see below). The standard procedure of subcellular fractionation was performed as previously described [32,33]. In brief, both the left and right retinae were washed gently with ice-cold PBS and processed for subcellular protein fractionation according to the manufacturer's protocol. The RBP levels were analyzed in the cytoplasmic extract (Ce), membrane extract (Me), soluble nuclear extract (Ne), and chromatin-bound nuclear extract (Cbe) fractions by immunoblotting.

Transmission Electron Microscopy (TEM)
TEM of the retinae of rd10, as well as that of age-matched WT, mice were performed using standard protocols [32,33]. Briefly, retinae were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 h followed by washing in the buffer for another 24 h. These samples were then incubated in 1% osmium tetroxide (OsO 4, in 0.2 M phosphate buffer) for 3 h, washed twice in distilled water, and dehydrated using ascending alcohol concentrations (i.e., 25%, 35%, 50%, 70%, 85%, 95%, 100%; each step for 5 min). Dehydrated blocks were incubated in propylene oxide followed by a subsequent 20 min incubation in a 1:1 mixture of epon (47.5% glycidether, 26.5% dodenylsuccinic acid anhydride, 24.5% methylnadic anhydride, and 1.5% Tris (dimethyl aminomethyl phenol) and propylene oxide. The samples were then incubated in epoxy resin for 1 h at room temperature followed by polymerization (28 • C for 8 h, 80 • C for 2.5 h, and finally at room temperature for 4 h). Ultra-thin sections (70 nm) were mounted on grids, contrast-enhanced with uranyl acetate and lead citrate, and examined using a Philips CM10 transmission electron microscope equipped with a Morada digital camera as already described [32,33].

Statistics
We used densitometric analysis on the protein bands on the Western blots by using Adobe Photoshop, representing the relative band intensity of the test proteins normalized with tubulin levels. Graphpad prism was used to conduct unpaired Student's t-tests for comparisons between two sample groups. Values were expressed as mean ± standard error of the mean (SEM) from three independent experiments. The asterisks (*) denote significant differences (* p < 0.05, ** p < 0.01). The (#) denotes not significant.

Increased Immunoreactivity and Protein Expression Levels of Calcium-Binding/Buffering Chaperones and ER Stress Markers in the rd10 Retina before any Noticeable Photoreceptor Loss at P17
Photoreceptor degeneration in the rd10 retina starts at 17-18 days after birth [11,13,14,21]. Consistent with the above studies, there was no obvious difference detected in the overall thickness measurements of the outer nuclear layer (ONL) of rd10-17 (see white scale bar), and any degenerative changes were hardly noticeable morphologically at this point when compared with the wild-type controls ( Figure 1a) by light microscopy. At P19, photoreceptor loss became apparent by detectable thinning of the ONL (Figure 1a). At P26, the thickness of the ONL was dramatically reduced due to a massive loss of photoreceptors (Figure 1a). At this stage, around 70-80% of the rod and at least 60-70% of the cone photoreceptor cells were already lost [21,22]. At P50, nearly all the photoreceptors from ONL had degenerated, while the inner retinal layers (INL and GCL) were barely affected ( Figure 1a). In contrast, no neurodegeneration was detected and there was no difference in the staining intensity and pattern of immunoreactivity of any of the test markers (used in this study) among the controls (WT) from P17, P19, P26, and P50.
Considering an overall calcium imbalance as an outcome of the Pde6b gene defect, we anticipated the dysregulation of Ca 2+ -associated proteins in photoreceptors at rd10-P17 and P26. In order to confirm this, we stained for the Ca 2+ binding protein calreticulin, which is involved in Ca 2+ buffering and storage. We found an increased calreticulin immunoreactivity at rd10-P17 and P26 in photoreceptor layers. Interestingly, we also observed increased immunostaining in the somata of the INL and GCL from rd10-P17 and P26 compared with WT-P26 and P50 (Figure 1b, quantification c), indicating that downstream targets are also affected. In addition, we observed increased calreticulin immunoreactivity in rod bipolar cells of rd10-P17 and P26 and even at P50, when the photoreceptors were almost completely lost ( Figure 1b, lower panel, quantification c). We used PKCα as a marker to detect rod bipolar cells, which are postsynaptic to rods. Their loss was apparent at the later stages of rd10 neurodegeneration [34,35]. These results were consistent with the Western blot analysis of the calreticulin protein performed on rd10 retinal lysates (Figure 1i, quantification j). Subsequently, we examined the immunoreactivity patterns of two other Ca 2+ buffering chaperones, PDI and pPERK. pPERK was also slightly increased in rod bipolar cells and photoreceptor inner segments (below the ONL) (Figure 1d, see also Table S2 for semi-quantitative analysis), while PDI was expressed more strongly in the processes running vertically through the retina, most likely originating from the Müller cells (the major retinal glia cell type) at rd10-P26 (Figure S1a, see also Table S2 for semi-quantitative analysis) in rd10 mice. Taken together, these results suggest alterations in Ca 2+ homeostasis already at an early stage before any obvious/major photoreceptor loss.
Next, we examined whether the ongoing Ca 2+ imbalance/altered Ca 2+ homeostasis is associated with increased ER stress levels. For this purpose, we used antibodies against the ER chaperones SigR1, GRP78/Bip, and GADD-153 (Figure 1e,f,h, quantification g). Whilst only a low level of SigR1 immunoreactivity was observed in WT retina, we found significant expression of SigR1 in the photoreceptor inner segments and in INL and GCL somata at rd10 P17, and also in photoreceptor somata at P26 (Figure 1e, quantification g). Immunoreactivity for GRP78/Bip and GADD-153 was only mildly increased (Figure 1f-h, quantification g, see also Table S2 for semi-quantitative analysis). GRP78/Bip was increased in the ganglion cell layer (GCL), suggesting chronic ER stress in these cells. Consistent with the immunohistochemical results, Western blotting analysis revealed significantly increased levels of GRP78/Bip, PDI, and calreticulin in both the rd10 retinae at P17 and P26 compared with the wild-type (WT) at P26, while pPERK and SigR1 protein levels were found to be significantly increased only at P26 of rd10 retinae (Figure 1i, quantification j).

Early Autophagy Alterations in the rd10 Retina
Autophagy is known to be tightly linked to the ER and decisive in preventing neurodegeneration mediated by ER stress [36,37]. Therefore, we hypothesized that altered ER-Ca 2+ homeostasis in the rd10 retina might lead to alterations in the autophagy process. As expected, immunoreactivities for the autophagy adaptor protein p62 and the autophagosome marker LC3 were significantly increased in the INL and the GCL of rd10-P17, and to a lesser extent of rd10-P26, compared with WT-P26 (Figure 2a,b; see also Table S2 for semi-quantitative analysis). In addition, several cells of rd10-P17 mice harbored cytoplasmic accumulations of p62-positive material/inclusions (Figure 2a, arrows, arrowhead). There was a significantly increased level of LC3 at rd10-P17, and a slight increase at P26; however, LC3 levels were not increased further at P50 (Figure 2b). Consistent with the immunolabelling results, immunoblot analysis revealed an overall increase in levels of p62 and increased levels of both LC3I [36] and LC3II in rd10-P17, as well as rd10-P26 retinal lysates (Figure 2d; quantification e; WT-P26 n = 3, rd10-P17 n = 4, rd10-P26 n = 3). Increased levels of both LC3I and lipidated LC3II (not shown) and altered levels of the ratio between LC3II/LC3I were suggestive of altered autophagy, most likely at both the initial (autophagosome formation/maturation) and final steps (autophagosome fusion to lysosomes) [38][39][40]. Neurodegeneration along with marked autophagy impairment in photoreceptors was later confirmed by the presence of large abnormal cytoplasmic autophagic vacuoles filled with membranous and granular debris in photoreceptor inner segments (IS) and somata in the ONL of rd10 mice at P19 (Figure 2f) and at P22 (Figure 2g) examined by electron microscopy. Such vacuoles were absent from WT controls (not shown). Taken together, these data show that signs of ER stress and defective autophagy are already manifested in photoreceptor and non-photoreceptor cell populations in the rd10 retina at early stages of photoreceptor degeneration.

Abnormal Cytoplasmic Aggregation of pTDP-43 and Matrin 3 at Early Stages of rd10 Retina Degeneration
ER-Ca 2+ dynamics can efficiently regulate autophagy as well as RBP homeostasis; on the other hand, aberrant ER-Ca 2+ may serve as a major driver for RBPs' alterations including TDP-43-mediated neuronal toxicity [41,42]. Similarly, the effective turnover/clearance of many disease-associated RBPs such as TDP-43 and FUS is regulated by the ubiquitinproteasome system (UPS) and autophagy [43,44]. Thus, we aimed to determine whether ongoing ER-Ca 2+ autophagy defects are associated with altered distribution of key RBPs linked to neurodegenerative disease, in particular TDP-43, FUS, and Matrin 3. These RBPs normally reside in the nucleus. In pathological/neurodegenerative conditions such as amyotrophic lateral sclerosis/frontotemporal lobar degeneration (ALS/FTLD), they often accumulate in the cytoplasm of affected neurons [45][46][47]. Using DAB immunohistochemistry, we found globular cytoplasmic pTDP-43 aggregates already present at P17 throughout the retinal cell layers including many photoreceptors, INL cells, and ganglion cells (Figure 3a; see also Table S2 for semi-quantitative analysis). pTDP-43 aggregates were also evident at considerable numbers at P26, but there was no further increment regarding their number nor their staining intensity (Figure 3a, right panel, see also Table S2 for semi-quantitative analysis) compared with P17. Immunofluorescence using the same antibody showed a higher sensitivity and revealed the accumulation of pTDP-43 in more than one-third of the photoreceptors (Figure 3b) at P17 and nearly 40% at P19 in the ONL, when the acute degeneration of the photoreceptors was the most pronounced (not shown). In line with the results obtained from the sections stained with DAB-immunohistochemistry mentioned above, pTDP-43 aggregation did not increase further at P26 nor at P50 (not shown) compared with P17. Interestingly, both DAB and immunofluorescence labelling consistently depicted pTDP-43 immunoreactive inclusions also present in cells at the outer margin of the inner nuclear layer. We recognized these cells as horizontal cells by using co-immunolabelling against the CabP antibody (Figure 3c).
The expression of TDP-43 is tightly regulated by another closely related RBP, Matrin 3 [48]. We found a mild cytoplasmic and stronger nuclear accumulation of Matrin 3 in rd10 mouse ONL, INL, and GCL cells at rd10-P17 and P26 (Figure 3d, arrows). Again, immunofluorescence labeling using the same antibody was very sensitive and revealed an altered pattern of Matrin 3 immunoreactivity, with increased granular nuclear accumulation of Matrin 3 in many cells (Figure 3e, arrows, right panel); on the other hand, several adjacent cells showed reduced levels of nuclear Matrin 3 immunoreactivity (Figure 3e, red arrowheads). Interestingly the photoreceptors showed an overall increase in the nuclear accumulation of Matrin 3 (arrows) in rd10-P17 compared with WT-P17 (Figure 3f, arrows). These results were further verified by Western blot analysis of subcellular fractions obtained from the retinal lysates (Figure 3g, red arrowheads; a pool of n = 3 WT-P17 and n = 3 rd10-P17, and

Aggregation of FET Family Proteins (FUS, EWRS1, and TAF15) in rd10 Retina
Members of the FET protein family (FUS, EWSR1, and TAF15) bind to RNA/DNA and regulate transcription, RNA processing, and other aspects of RNA/DNA homeostasis [49][50][51][52]. FET proteins are involved in several neurodegenerative diseases as they can also form toxic aggregates, and perturb protein homeostasis, thus driving neurodegeneration [49,52]. We observed globular cytoplasmic FUS protein accumulation in both photoreceptors and many cells of the INL (Figure 4a, white arrows). Focal intra-nuclear FUS-positive accumulations, as well as strongly labeled FUS-positive condensed/pyknotic nuclei (Figure 4a, black arrows), were also evident. Similar to the pattern of FUS immunoreactivity, other FET family member proteins such as hnRNPA1, hnRNPA2B1, and EWSR1 often showed focal intranuclear accumulation (Figure 4b-d, black arrows). hnRNPA1 and EWSR1, however, did not show significant cytoplasmic accumulation at P17, but at P26, several cytoplasmic globular aggregates were visible in photoreceptors and INL cells (Figure 4b (Figure 4e, white arrows), were evident in rd10 mice at P17, P19, and P26. In addition to this, large FUS-immunoreactive aggregates were also evident in GCL cells at rd10-P17 (not shown) and P26 (Figure 4e, white arrow, lower panel). The formation of SDS-resistant FUS aggregates in the rd10 retina was further confirmed by using a standard filter trap assay (Figure 4f). Consistent with this, subcellular fraction analysis using Western blots of retinal lysates further confirmed the cytoplasmic mislocalization (red arrowhead) of FUS, hnRNPA1, and TAF15 in rd10-P17 and rd10-P26 retinae (pool of n = 3 WT-P26, n = 3 rd10-P17, and n = 3 rd10-P26, Figure 4g). revealed globular cytoplasmic accumulation (white arrows in (a-d)) in photoreceptors of the ONL and many cells of the INL. Focal intranuclear accumulations (black arrows), as well as condensed/pyknotic nuclei, were also evident. A single representative section from every genotype (n = 3 WT-P26, n = 3 WT-P50, n = 3 rd10-P17, n = 3 rd10-P26, and n = 3 rd10-P50) is depicted.

Formation of RNA Stress Granules (SGs) in rd10 Retina
The aggregation of the above-mentioned RBPs mostly proceeds through the SG pathway and might thus serve as a seed for further pathological aggregates, if persistent [46,47,[53][54][55]. Therefore, we asked whether the accumulations of the RBPs studied were accompanied by SG formation. We found small globular cytoplasmic aggregations of the widely studied SG protein Tia1 in photoreceptors and INL cells of rd10-P17 and P26 mice (Figure 5a, white arrows), together with focal intranuclear accumulation (Figure 5a, black arrows). Interestingly, Tia1-positive SG accumulations were abundant especially at early stages of retinal degeneration (P17), and they appeared less frequently at a later stage (P26) (Figure 5a, right panel P26). Co-immunolabelling using a combination of Tia1 and FUS or TDP-43 antibodies revealed a clear pattern of sequestration of Tia1 with FUS and TDP-43 within these aggregates (Figure 5b, arrows). Consistent with this, subcellular fraction analysis using Western blots of retinal lysates further confirmed the cytoplasmic accumulation of Tia1 (red arrowheads) at P17 and P26 (pool of n = 3 each of WT-P26, rd10-P17, and rd10-P26, Figure 5c). These results further confirm the notion that RBP aggregates proceed through the SG pathway and that aberrant SGs containing RBPs can be considered to be an early pathological event in the aggregation of RBPs.

Discussion
In the commonly used rd10 mouse model of retinitis pigmentosa, a mutation in the Pde6 gene leads to photoreceptor degeneration and consequent blindness [11][12][13][14][15]. Milder and slower degeneration in certain second and third-order neurons also occurs. As Pde6 is exclusively expressed in rod photoreceptors, this degeneration must be a secondary effect, e.g., as a consequence of the lack of synaptic input by the photoreceptors [11][12][13][14][15]21]. However, the pathomechanisms of the degeneration of non-photoreceptor neuronal cell types in RP are poorly understood. Photoreceptor degeneration in the rd10 retina starts at 17-18 days after birth [11][12][13][14][15]21]. At P17, all cell layers still appear anatomically unchanged compared with age-matched WT retina (Figure 1), and light responses can be recorded on the level of the electroretinogram or individual ganglion cells [14,56]. Interestingly, at P17, we could already find the expression of several markers commonly observed in other types of neuronal degeneration, not only in photoreceptors but also in cells of the INL and GCL. We found (a) increases in the Ca 2+ buffering chaperones calreticulin, PDI, and pPERK, together with the ER chaperones SigR1 and GRP78/Bip; (b) altered expression of the autophagy proteins p62 and LC3, together with the accumulation of autophagic vacuoles; (c) cytoplasmic and nuclear aggregation of RBPs such as pTDP-43 and FET-family RBPs (see also schematic representation Figure 6). In the early stage, only rod photoreceptors die, followed by cones later in the process. In line with this, we found that the two cell types that receive direct input from rods and which display considerable loss at later stages of retinal degeneration [34,35], i.e., the rod bipolar cells and the horizontal cells (see, e.g., Figures 1 and 3), express the markers summarized above. These results suggest that deleterious effects due to altered Ca 2+ homeostasis and ER stress initiated in rods lead to similar alterations in interconnected cell types. From this point of view, it appears logical that these cell populations upregulate levels of neuroprotective ER chaperones. In fact, SigR1 is extensively studied for its neuroprotective role in retinal cells [57][58][59][60].
Above-threshold Ca 2+ imbalance and ER stress that cannot be managed by the abovementioned factors should eventually lead to the failure of cellular proteostasis networks including protein quality control/autophagy pathways, resulting in misfolded protein aggregation and associated neurotoxicity (see also schematic representation Figure 6). This has been observed in major chronic neurodegenerative disorders including Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) [25,61,62]. Altered proteostasis has also been previously discussed as a factor contributing to the spread of degenerative pathology in RP retina cell populations [15,21]. The results of the present study add credibility to this concept. They add the surprising observation that abnormal protein aggregates can already be observed at the early stages of neurodegeneration, not only in photoreceptors but also in non-photoreceptor cell types, and that they include aggregates of RBPs-most prominently of TDP-43, an important player in the pathophysiology of several major neurodegenerative diseases including ALS/FTLD [45,52]. Additionally, toxic modifications of these protective proteins caused by chronic cellular stress conditions could contribute to progressive neurodegenerative phenotypes as seen in rd10 retina [15,21,22,63]. RBPs regulate several crucial aspects of neuronal gene expression including splicing regulation, mRNA transport, and modulation of mRNA translation and decay. In addition, RBPs are known to regulate proteostasis and autophagy at multiple steps [45]; in turn, the efficient turnover of several disease-associated RBPs including TDP-43, FUS, and SGs is regulated by UPS and autophagy [43,44]. One of the intriguing aspects observed here is that the build-up of RBP aggregates is happening rather early and quickly, along with the buildup of altered ER-Ca 2+ homeostasis and ER stress. Thus, it appears reasonable to assume that the ongoing chronic cellular stress conditions may quickly lead to defects in the nucleus regarding cytoplasmic shuttling and thus to impaired RBP homeostasis [45][46][47]50,53].
Altered RBP homeostasis and defective proteostasis/autophagy are key mechanisms triggering neurodegeneration in many neurodegenerative diseases including ALS [45][46][47]50,53]. We observed both altered autophagy and RBP aggregations at early stages throughout the retinal cell layers, even though the Pde6b gene defect selectively affects rod photoreceptor cells. Moreover, the Pde6b gene does not primarily target RBP metabolism. The RBPs studied here, including TDP-43 and FET proteins such as FUS and EWSR1, are particularly susceptible to aggregation due to the presence of both RNA-binding domains and prionlike domains enriched in uncharged polar amino acids (such as asparagine, glutamine, and tyrosine), which contribute strongly to aggregate formation under stressful stimuli including ER-Ca 2+ imbalance, ER-stress, and autophagy impairment [50,51,55,64]. These findings are also reminiscent of various age-related neurodegenerative diseases such as AD, PD, HD, and prion diseases, where specific subsets of neurons are affected and the disease-causing protein aggregates initially appear in a restricted distribution and only later spread throughout the CNS [42]. We observed a close association of RBP build-up with stress granule formation in affected neuronal cells, especially at an early stage (P17) of retinal degeneration in rd10 mice, suggesting that these aggregates arise de novo in these cells.
Further studies will be required to determine how the Pde6b gene mutation leads to this cascade of events. Regardless of the initial events, considering the hierarchical cluster of heterogeneous types of neurons and interconnected, interdependent units, we cannot exclude the possibility that a prion-like seeding and spreading of the above-mentioned RBPs could be implicated further downstream, similar to the spreading of RBP aggregates in other neurodegenerative diseases [65,66].
We conclude that-owing to the clear separation of cell types affected by primary and secondary degeneration-the retina provides an exquisite model to study neuronal degeneration processes, in particular as many similarities were found to processes described for other neurodegenerative disorders. Progressive neurodegeneration in the rd10 mouse retina is associated with early disturbances of proteostasis and autophagy and with abnormal cytoplasmic RBP aggregation and stress granule formation. These processes are already triggered in the very early phases of retinal degeneration (see also schematic representation Figure 6).